This invention relates to compounds which are effective in inhibition of angiogenesis, and accordingly can be used instead of sulfated polysaccharides such as heparin in preventing restenosis, accelerating wound healing, and in inhibiting tumor cell metastasis.
The use of sulfated polysaccharides in the inhibition of angiogenesis and in the treatment of disorders and conditions associated with angiogenesis has been previously disclosed. Thus, International Patent Application No. PCT/GB95/00515 (WO 95/24907), the contents of which are incorporated herein by reference, discusses the use of heparin and other sulfated polysaccharides such as pentosan polysulfate and dextran sulfate in treatment of these disorders and conditions, and discloses the use of another sulfated polysaccharide, laminarin sulfate, which exhibits only about 30% of the anti-coagulant activity of heparin, in preventing restenosis by the inhibition of vascular smooth muscle cell proliferation, in accelerating wound healing by activating the release of active growth factors stored in the extra-cellular matrix, and for inhibiting tumor cell metastasis by inhibition of heparanase activity.
International Patent Application No. PCT/AU95/00350 (WO 95/34595) discloses a class of antiviral compounds comprising a dendrimer such as a polyamidoamine or polylysine dendrimer having a plurality of terminal groups, wherein at least one of the terminal groups has an anionic- or cationic-containing moiety bonded or linked thereto, particularly a sulfonic acid-containing, a carboxylic acid-containing, or a trimethyl-ammonium-containing moiety.
The present invention provides the use of polyionic materials formed by linking ionic groups to a linear non-carbohydrate polymer or a dendritic polymer in the inhibition of angiogenesis and in the treatment of related disorders and conditions.
According to the present invention, there is provided a method of prophylactic or therapeutic inhibition of angiogenesis in a human or non-human animal patient, which comprises administration to the patient of an effective amount of a compound selected from:
(i) linear, non-carbohydrate polymers having a plurality of side chain groups wherein at least one of said side chain groups has an anionic- or cationic-containing moiety bonded or linked thereto; and
(ii) dendrimers having a plurality of terminal groups wherein at least one of said terminal groups has an anionic- or cationic-containing moiety bonded or linked thereto.
Particularly preferred compounds for use in the method of the present invention are linear polymers having sulfonic acid-containing moieties, carboxylic acid-containing moieties, phosphoric or phosphonic acid-containing moieties, boronic acid-containing moieties, neuraminic or sialic acid-containing moieties or moieties containing neuraminic or sialic acid modified in the 4- or other position thereof, linked to side chain groups thereof, and dendrimers having sulfonic acid-containing moieties, carboxylic acid-containing moieties, phosphoric or phosphonic acid-containing moieties, boronic acid-containing moieties, neuraminic or sialic acid-containing moieties or moieties containing neuraminic or sialic acid modified in the 4- or other position thereof, linked to terminal groups thereof.
The compounds used in the method of this invention are referred to herein are linear polyionic polymers or polyionic dendrimers, respectively, and these terms are used throughout this specification and the claims which follow to include not only the polymers or dendrimers per se, but also their pharmaceutically or veterinarily acceptable salts, for example the alkaline metal or alkaline earth metal salts such as the sodium, potassium or calcium salts.
Preferred compounds used in accordance with the present invention include (i) linear polyionic polymers of the general formula I: 
wherein:
R is a non-carbohydrate monomer unit forming a linear polymer backbone;
X is an optional linking group on the side chain groups of monomer units R; and
A is an anionic-containing moiety; and (ii) polyionic dendrimers of the general formula II: 
wherein:
I is an initiator core;
Z is an interior branching unit;
n is an integer which represents the number of generations of the dendrimer; and
A is an anion-containing moiety which may be linked to interior branching unit Z through an optional linking group X.
In accordance with the present invention, the preferred linear polyionic polymers are polyanionic materials formed by the conjugation of anionic moieties (A) to a linear non-carbohydrate polymer backbone (made up of a plurality of monomer units R), optionally through linking groups (X). The resultant polyanionic linear polymers have a weight range distribution of repeating units to give a desired median range of molecular weight distribution. Desirably, the median range of molecular weight distribution is from 1,000 to 1,000,000, preferably from 10,000 to 600,000.
The monomer unit R is preferably an amino or amide moiety, more preferably an amino acid moiety. A particularly preferred monomer unit is a lysine moiety. Poly-L-lysines having various molecular weight ranges are available commercially from Sigma Chemical Company.
The anionic moiety A can be linked to reactive side chain groups on the linear polymer backbone either directly or via a variety of functional linking groups X such as, but not limited to, esters, amides, ethers, thioethers, amines, ureas, thioureas, carbamates and carbonates.
The optional linking group X may also act as a spacer between the polymer and the anionic moiety A, and may consist of an alkyl chain (optionally substituted or branched), an alkoxy, polyalkoxy, alkylthio or polyalkylthio chain (optionally substituted), or an alkenyl, multiple alkenyl, alkynyl or multiple alkynyl chain (optionally substituted). Suitable spacer chains include groups of the formula xe2x80x94(CH2)nxe2x80x94Zxe2x80x94(CH2)nxe2x80x94, wherein Z is xe2x80x94CH2xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, and n is an integer of from 1 to 15.
Dendrimers are macromolecular highly branched compounds formed by reiterative reaction sequences starting from an initial, core molecule with successive layers or stages being added in successive xe2x80x9cgenerationsxe2x80x9d to build up a three-dimensional, highly ordered polymeric compound. Dendrimers are characterised by the following features: I an initiator core(I) which may have one or more reactive sites and be point-like or of significant size so as to effect the final topology of the dendrimer; ii layers of branched repeating units (Z) attached to the initiator core; iii functional terminal groups (such as anionic moieties A) attached to the surface of the dendrimer, optionally through linking groups (such as linking groups X described above). The present invention uses dendritic structures as frameworks for the attachment of ionic moieties; the invention is not limited to the spherical dendrimers described in detail herein but can be based on any dendritic structure. The variety of dendrimers in both shape and constitution are well known to persons skilled in the art.
The preparation of dendrimers is well known, and is described by way of example in U.S. Pat. Nos. 4,289,872 and 4,410,688 (describing dendrimers based on layers of lysine units), as well as U.S. Pat. Nos. 4,507,466, 4,558,120, 4,568,737 and 4,587,329 (describing dendrimers based on other units including polyamidoamine or PAMAM dendrimers). The dendrimers disclosed in these US patents are described as being suitable for uses such as surface modifying agents, as metal chelating agents, as demulsifiers or oil/water emulsions, wet strength agents in the manufacture of paper, and as agents for modifying viscosity in aqueous formulations such as paints. It is also suggested in U.S. Pat. Nos. 4,289,872 and 4,410,688 that the dendrimers based on lysine units can be used as substrates for the preparation of pharmaceutical dosages.
International Patent Publications Nos. WO 88/01178, WO 88/01179 and WO 88/01180 disclose conjugates in which a dendrimer is conjugated or associated with another material such as a carried pharmaceutical or agricultural material. In addition, International Patent Publication No. WO 95/24221 discloses dendritic polymer conjugates composed of at least one dendrimer in association with a carrier material which can be a biological response modifier, and optionally a target director. These patent publications together with the U.S. patents mentioned above contain a broad disclosure of various dendrimers and processes for the preparation thereof, and the disclosure of each of these publications is incorporated herein by reference.
The term xe2x80x9cdendrimerxe2x80x9d as used herein is to be understood in its broadest sense, and to include within its scope all forms and compositions of these dendrimers as disclosed in Patent Publications Nos. WO 88/01178, WO 88/01179 and WO 88/01180. The term also includes linked or bridged dendrimers as disclosed in these patent publications.
The preferred dendrimers of the present invention comprise a polyvalent core covalently bonded to at least two dendritic branches, and preferably extend through at least two generations. Particularly preferred dendrimers are polyamidoamine (PAMAM) dendrimers, PAMAM (EDA) dendrimers and polylysine dendrimers.
In accordance with the present invention, at least one, and preferably a substantial number, of the side chain groups on the linear polymer or terminal groups on the surface of the dendrimer has an anionic- or cationic-containing moiety covalently bonded thereto. The side chains of the linear polymer or branches of the dendrimer may terminate in amino groups or other functional reactive groups such as OH, SH, or the like, which subsequently can be reacted with the anionic or cationic moieties. Where the side chain groups of the linear polymer or terminal groups of the dendrimer are amine groups, the anionic- or cationic-containing moiety may be linked to the dendrimer by a variety of functional groups including amide and thiourea linkages. Preferred anionic- or cationic-containing moieties which may be bonded to the side chain groups of the linear polymer or terminal groups of the dendrimer include sulfonic acid-containing moieties, carboxylic acid-containing moieties (including neuraminic and sialic acid-containing moieties and modified neuraminic and sialic acid-containing moieties), boronic acid-containing moieties, phosphoric and phosphonic acid-containing moieties (including esterified phosphoric and phosphonic acid-containing moieties) and trimethylammonium-containing moieties.
Suitable anionic- and cationic-containing moieties which may be bonded or linked to the amino or other side chain or terminal groups include, by way of example, the following groups (in which n is zero or a positive integer, more particularly n is zero or an integer of from 1 to 20):
xe2x80x94NH(CH2)nSO3xe2x88x92 xe2x80x94(CH2)nSO3xe2x88x92 xe2x80x94Ar(SO3xe2x88x92)n 
xe2x80x94CH2CH(SO3xe2x88x92)COOH xe2x80x94CH(SO3xe2x88x92)CH2COOH xe2x80x94ArX(CH2)nSO3xe2x88x92 X=O, S, NH 
xe2x80x94ArXP(xe2x95x90O)(OR)2  X=O, CH2, CHF, CF2 R=alkyl, aryl, H, Na,
xe2x80x94ArXP(xe2x95x90O)(OR1)(NR2R3) X=O, CH2, CHF, CF2 R1=alkyl, aryl, H, Na R2, R3=alkyl, aryl
xe2x80x94Ar[P(xe2x95x90O)(OR)2]n R=alkyl, aryl, H, Na n=1-3
xe2x80x94Ar[B(OH)2]n n=1-3 xe2x80x94Ar[COOH]n n=1-3 
In addition to the above, various neuraminic or sialic acid-containing moieties or modified neuraminic or sialic acid-containing moieties may be bonded or linked to the side chain or terminal groups in accordance with this invention. These moieties include the various N- and O-substituted derivatives of neuraminic acid, particularly N- and O-acyl derivatives such as N-acetyl, O-acetyl and N-glycolyl derivatives, as well as moieties in which the neuraminic acid group is modified, particularly by substitution in the 4-position, with an amino, amido, cyano, azido or guanidino group.
The anionic or cationic linear polymers and dendrimers of this invention may be prepared by standard chemical methods which are well known to persons skilled in this art. Suitable methods are described by way of the example in Examples below.
As previously described, the anionic or cationic linear polymers and dendrimers of the present invention have been found to inhibit angiogenesis. Accordingly, the method of the present invention includes inhibition of angiogenesis in a patient, treatment of conditions where growth of new blood vessels is involved such as chronic inflammation, diabetic retinopathy, psoriasis and rheumatoid arthritis, as well as treatment of related disorders and conditions including, but not limited to, prevention of restenosis by inhibition of vascular smooth muscle cell proliferation, acceleration of wound healing by activation of the release of active growth factors stored in the extracellular matrix, and inhibition of tumor cell metastasis by inhibition of angiogenesis.
Thus, in another aspect the present invention provides a pharmaceutical or veterinary composition for prophylactic or therapeutic inhibition of angiogenesis in a human or non-human animal patient, which comprises an anionic or cationic linear polymer or dendrimer as broadly described above, in association with at least one pharmaceutically or veterinarily acceptable carrier or diluent.
The formulation of such compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington""s Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.
In yet another aspect, this invention provides the use of an effective amount of an anionic or cationic linear polymer or dendrimer as broadly described above in the prophylactic or therapeutic treatment of, or in the manufacture of a medicament for prophylactic or therapeutic treatment of a human or non-human animal patient by inhibition of angiogeneis.
A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, inhalation, transdermal or parenteral (e.g. subcutaneous, intramuscular and intravenous) routes. Formulations for oral administration include discrete units such as capsules, tablets, lozenges and the like. Other routes include intrathecal administration directly into spinal fluid, direct introduction such as by various catheter and balloon angioplasty devices well known to those of ordinary skill in the art, and intraparenchymal injection into targeted areas.
The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing the active component into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active component into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active component which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in polyethylene glycol. Among the acceptable vehicles and solvents that may be employed are water, Ringer""s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The active component may also be formulated for delivery in a system designed to administer the active component intranasally or by inhalation, for example as a finely dispersed aerosol spray containing the active component.
Other delivery systems can include sustained release delivery systems. Preferred sustained release delivery systems are those which can provide for release of the active component of the invention in sustained release pellets or capsules. Many types of sustained release delivery systems are available. These include, but are not limited to: (a) erosional systems in which the active component is contained within a matrix, and (b) diffusional systems in which the active component permeates at a controlled rate through a polymer. In addition, a pump-based hardware delivery system can be used, some of which are adapted for implantation.
The active component is administered in prophylactically or therapeutically effective amounts. A prophylactically or therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons.
Generally, daily oral doses of active component will be from about 0.01 mg/kg per day to 1000 mg/kg per day. Small doses (0.01-1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localised delivery route) may be employed to the extent patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
The active component according to the invention may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for:
(a) oral administration, external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;
(b) parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced into the udder via the teat;
(c) topical application, e.g. as a cream, ointment or spray applied to the skin; or
(d) intravaginally, e.g. as a pessary, cream or foam.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
Further features of the present invention will be apparent from the following Examples which are included by way of illustration, not limitation of the invention. In the following Examples, PAMAM dendrimers refer to polyamidoamine dendrimers based on an ammonia core as detailed in U.S. Pat. Nos. 4,507,466, 4,558,120, 4,568,737 and 4,587,329; PAMAM (EDA) dendrimers refer to polyamidoamine dendrimers based on an ethylene diamine core; and BHAlysxlysylysz dendrimers refer to polylysine unsymmetrical dendrimers based on a benzhydrylamine core and lysine branching units as described in U.S. Pat. Nos. 4,289,872 and 4,410,688. The polyamidoamine dendrimers PAMAM 1.0, PAMAM 2.0, PAMAM 3.0, PAMAM 4.0, PAMAM 5.0 or higher generation, PAMAM 4.0 (EDA), and the polylysine dendrimers BHAlyslys2, BHAlyslys2lys4, BHAlyslys2lys4lys8 and BHAlyslys2lys4lys8lys16, BHAlyslys2lys4lys8lys16lys32, BHAlyslys2lys4lys8lys16lys32lys64, or higher generations prepared as described in U.S. Pat. Nos. 4,289,872, 4,410,688, 4,507,466, 4,558,120, 4,568,737 and 4,578,239 and International Patent Publications Nos. WO 88/01178, WO 88/01179, WO 88/01180 and WO 95/24221 referred to above.